Infection Of Human Cells Research Articles

Identification of UL69 Gene and Protein in Cytomegalovirus-Transformed Human Mammary Epithelial Cells.

A growing body of evidence addressing the involvement of human cytomegalovirus (HCMV) in malignancies had directed attention to the oncomodulation paradigm. HCMV-DB infected human mammary epithelial cells (HMECs) in culture showed the emergence of clusters of rapidly proliferating, spheroid-shaped transformed cells named CTH (CMV-Transformed HMECs) cells. CTH cells assessment suggests a direct contribution of HCMV to oncogenesis, from key latent and lytic genes activating oncogenic pathways to fueling tumor evolution. We hypothesized that the presence of HCMV genome in CTH cells is of pivotal importance for determining its oncogenic potential. We previously reported the detection of a long non-coding (lnc) RNA4.9 gene in CTH cells. Therefore, we assessed here the presence of UL69 gene, located nearby and downstream of the lncRNA4.9 gene, in CTH cells. The HCMV UL69 gene in CTH cells was detected using polymerase chain reaction (PCR) and sequencing of UL69 gene was performed using Sanger method. The corresponding amino acid sequence was then blasted against the UL69 sequence derived from HCMV-DB genome using NCBI Protein BLAST tool. A 99% identity was present between the nucleotide sequence present in CTH cells and HCMV-DB genome. UL69 transcript was detected in RNA extracts of CTH cells, using a reverse transcription polymerase chain reaction (RT-PCR) assay, and pUL69 protein was identified in CTH lysates using western blotting. Ganciclovir-treated CTH cells showed a decrease in UL69 gene detection and cellular proliferation. In CTH cells, the knockdown of UL69 with siRNA was assessed by RT-qPCR and western blot to reveal the impact of pUL69 on HCMV replication and CTH cell proliferation. Finally, UL69 gene was detected in breast cancer biopsies. Our results indicate a close link between the UL69 gene detected in the HCMV-DB isolate used to infect HMECs, and the UL69 gene present in transformed CTH cells and tumor biopsies, further highlighting a direct role for HCMV in breast tumor development.

SERINC5 Can Enhance Proinflammatory Cytokine Production by Primary Human Myeloid Cells in Response to Challenge with HIV-1 Particles.

HIV-1 has to overcome physical barriers posed by host cell restriction factors (RFs) for efficient replication. Some RFs, including Trim5α and tetherin, trigger antiviral signaling in addition to directly impairing HIV replication. SERINC5 (S5) is an RF that is incorporated into HIV-1 particles to potently impair their infectivity and is efficiently antagonized by the viral pathogenesis factor Nef. Since effects of S5 on HIV-1 infectivity were mostly studied in reporter cell lines, we analyzed the effects of S5 during infection of primary HIV-1 target cells. In activated CD4+ T lymphocytes, virion incorporation of S5 only moderately impaired virion infectivity and was not associated with altered innate immune recognition. In contrast, in monocyte-derived macrophages, S5 virion incorporation potentiated the production of proinflammatory cytokines with very potent but donor-dependent effects on virion infectivity. Nef counteracted effects of S5 on both cytokine production and virion infectivity. Similar S5-induced cytokine production was observed in immature monocyte-derived dendritic cells. Notably, S5-mediated enhancement of cytokine production was not linked to the efficacy of productive infection and could be overcome by using vesicular stomatitis virus glycoprotein (VSV-G) but not infectivity restriction-insensitive HIV-1 Env for cell entry. Moreover, inhibiting entry of S5-negative HIV-1 ΔNef particles increased proinflammatory cytokine production comparably to virion incorporation of S5. Together, these results describe the sensitization of noninfectious HIV-1 particles to proinflammatory cytokine production by myeloid target cells as an additional and Nef-sensitive activity of S5. Moreover, the study reveals important cell-type and donor-dependent differences in the sensitivity of HIV target cells for antiviral effects of S5.IMPORTANCE SERINC5 (S5) is a host cell restriction factor (RF) that impairs the infectivity of HIV-1 particles in target cell lines. To assess the potential physiological relevance of this restriction, we assessed the effects of S5 on HIV-1 infection of relevant primary human target cells. We found that effects of S5 on infection of CD4+ T lymphocytes were negligible. In myeloid target cells, however, virion incorporation of S5 potently suppressed infectivity and promoted innate immune recognition of HIV-1 particles characterized by proinflammatory cytokine production. Both effects were not observed in cells of all donors analyzed, were exerted independently of one another, and were counteracted by the HIV-1 pathogenesis factor Nef. These results identify the sensitization of HIV-1 particles for innate immune recognition by myeloid target cells as a novel activity of S5 and emphasize the need to study RF function in the context of primary target cells and taking donor variabilities into account.

Analysis of DnaK Expression from a Strain of Mycoplasma fermentans in Infected HCT116 Human Colon Carcinoma Cells.

Several species of mycoplasmas, including Mycoplasma fermentans, are associated with certain human cancers. We previously isolated and characterized in our laboratory a strain of human mycoplasma M. fermentans subtype incognitus (MF-I1) able to induce lymphoma in a Severe Combined Immuno-Deficient (SCID) mouse model, and we demonstrated that its chaperone protein, DnaK, binds and reduces functions of human poly-ADP ribose polymerase-1 (PARP1) and ubiquitin carboxyl-terminal hydrolase protein-10 (USP10), which are required for efficient DNA repair and proper p53 activities, respectively. We also showed that other bacteria associated with human cancers (including Mycoplasma pneumoniae, Helicobacter pylori, Fusobacterium nucleatum, Chlamydia thrachomatis, and Chlamydia pneumoniae) have closely related DnaK proteins, indicating a potential common mechanism of cellular transformation. Here, we quantify dnaK mRNA copy number by RT-qPCR analysis in different cellular compartments following intracellular MF-I1 infection of HCT116 human colon carcinoma cells. DnaK protein expression in infected cells was also detected and quantified by Western blot. The amount of viable intracellular mycoplasma reached a steady state after an initial phase of growth and was mostly localized in the cytoplasm of the invaded cells, while we detected a logarithmically increased number of viable extracellular bacteria. Our data indicate that, after invasion, MF-I1 is able to establish a chronic intracellular infection. Extracellular replication was more efficient while MF-I1 cultured in cell-free axenic medium showed a markedly reduced growth rate. We also identified modifications of important regulatory regions and heterogeneous lengths of dnaK mRNA transcripts isolated from intracellular and extracellular MF-I1. Both characteristics were less evident in dnaK mRNA transcripts isolated from MF-I1 grown in cell-free axenic media. Taken together, our data indicate that MF-I1, after establishing a chronic infection in eukaryotic cells, accumulates different forms of dnaK with efficient RNA turnover.

Differential inflammatory response to Aspergillus flavus and Candida albicans infection in human retinal pigment epithelial cells: Role in treatment of endophthalmitis

ObjectiveFungal endophthalmitis is an emerging vision-threatening complication in tropical countries and the Retinal pigment epithelial cells (RPE) are said to play a major role in the retinal pathology. The aim of this study was to compare the immune response of Retinal pigment epithelial cells (RPE) challenged with A. flavus and C. albicans strains, isolated from patients with fungal endophthalmitis. Material and MethodsRetinal Pigment epithelial cells (ARPE-19) cells were infected with A. flavus and C. albicans, and gene expression were assessed for TLRs, immune-mediators, and matrix metalloproteinases (MMPs). ResultsWe observed a time-dependent gene expression of TLRs (TLR-2,-7 and -9); IL-8 and TNF-α in RPE cells challenged with A. flavus and C. albicans. Additonally, IL-6 (3.3 fold), IL-10 (15.2 fold), and IL-17 (5.6 fold) were significantly up-regulated only in cells infected with A. flavus. Additionally, MMP-9 gene expression was up-regulated in both A.flavus as well as C.albicans infected cells, while MMP- 2 gene expression was induced only in cells infected with C.albicans. A total of 9 upregulated differential expression of genes (DEGs) in A. flavus infected cells and 7 DEGs in C. albicans infected cells were used to construct Protein-protein interaction (PPI) network. ConclusionRPE cells induced a differential innate immune response depending on fungal species involved (A.flavus and C.albicans) and may provide clues for novel treatment targets and better prognosis.

The role of microRNAs in modulating SARS-CoV-2 infection in human cells: a systematic review.

MicroRNAs are gene expression regulators, associated with several human pathologies, including the ones caused by virus infections. Although their role in infection diseases is not completely known, they can exert double functions in the infected cell, by mediating the virus infection and/or regulating the immunity-related gene targets through complex networks of virus-host cell interactions. In this systematic review, the Pubmed, EMBASE, Scopus, Lilacs, Scielo, and EBSCO databases were searched for research articles published until October 22nd, 2020 that focused on describing the role, function, and/or association of miRNAs in SARS-CoV-2 human infection and COVID-19. Following the PRISMA 2009 protocol, 29 original research articles were selected. Most of the studies reported miRNA data based on the genome sequencing of SARS-CoV-2 isolates and computational prediction analysis. The latter predicted, by at least one independent study, 1266 host miRNAs to target the viral genome. Thirteen miRNAs were identified by four independent studies to target SARS-CoV-2 specific genes, suggested to act by interfering with their cleavage and/or translation process. The studies selected also reported on viral and host miRNAs that targeted host genes, on the expression levels of miRNAs in biological specimens of COVID-19 patients, and on the impact of viral genome mutations on miRNA function. Also, miRNAs that regulate the expression levels of the ACE2 and TMPRSS2 proteins, which are critical for the virus entrance in the host cells, were reported. In conclusion, despite the limited number of studies identified, based on the search terms and eligibility criteria applied, this systematic review provides evidence on the impact of miRNAs on SARS-CoV-2 infection and COVID-19. Although most of the reported viral/host miRNAs interactions were based on in silico prediction analysis, they demonstrate the relevance of the viral/host miRNA interaction for viral activity and host responses. In addition, the identified studies highlight the potential use of miRNAs as therapeutic targets against COVID-19, and other viral human diseases (This review was registered at the International Prospective Register of Systematic Reviews (PROSPERO) database (#CRD42020199290).

Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

To discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

#71: Single-cell RNA Sequencing Analysis of Zika Virus Infection in Human Stem Cell-Derived Neuroprogenitor Cells and Cerebral Organoids

Abstract Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. It is also unclear why congenital ZIKV disease was not observed prior to the recent epidemics in French Polynesia and the Americas, despite evidence that the Zika virus has actively circulated in parts of Africa and Asia since 1947 and 1966, respectively. Methods Due to advances in stem cell-based technologies, we can now model ZIKV infections of the central nervous system in human stem cell-derived neuroprogenitor cells and cerebral organoids, which recapitulate complex three-dimensional neural architecture. We apply Seq-Well—a simple, portable platform for massively parallel single-cell RNA sequencing—to characterize these neural models infected with ZIKV. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, thereby defining transcriptional features of both uninfected and infected cells. Results In neuroprogenitor cells, single-cell sequencing reveals that while uninfected bystander cells strongly upregulate interferon pathway genes, these are largely suppressed in cells infected with ZIKV within the same culture dish. In our organoid model, single-cell sequencing allows us to identify multiple cellular populations, including neuroprogenitor cells, intermediate progenitor cells, and terminally differentiated neurons. In this model of the developing brain, we identify preferred tropisms of ZIKV infection. Our data additionally reveal differences in cell-type frequencies and gene expression within organoids infected by historic and contemporary ZIKV strains from a variety of geographic locations. Conclusions These findings may help explain phenotypic differences attributed to the viruses, including variable propensities to cause microcephaly. Overall, our work provides insight into normal and diseased human brain development and suggests that both virus replication and host response mechanisms underlie the neuropathology of ZIKV infection.

Lung Epithelial Cell Transcriptional Regulation as a Factor in COVID-19-associated Coagulopathies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global pandemic. In addition to the acute pulmonary symptoms of coronavirus disease (COVID-19) (the disease associated with SARS-CoV-2 infection), pulmonary and distal coagulopathies have caused morbidity and mortality in many patients. Currently, the molecular pathogenesis underlying COVID-19–associated coagulopathies are unknown. Identifying the molecular basis of how SARS-CoV-2 drives coagulation is essential to mitigating short- and long-term thrombotic risks of sick and recovered patients with COVID-19. We aimed to perform coagulation-focused transcriptome analysis of in vitro infected primary respiratory epithelial cells, patient-derived bronchial alveolar lavage cells, and circulating immune cells during SARS-CoV-2 infection. Our objective was to identify transcription-mediated signaling networks driving coagulopathies associated with COVID-19. We analyzed recently published experimentally and clinically derived bulk or single-cell RNA sequencing datasets of SARS-CoV-2 infection to identify changes in transcriptional regulation of blood coagulation. We also confirmed that the transcriptional expression of a key coagulation regulator was recapitulated at the protein level. We specifically focused our analysis on lung tissue–expressed genes regulating the extrinsic coagulation cascade and the plasminogen activation system. Analyzing transcriptomic data of in vitro infected normal human bronchial epithelial cells and patient-derived bronchial alveolar lavage samples revealed that SARS-CoV-2 infection induces the extrinsic blood coagulation cascade and suppresses the plasminogen activation system. We also performed in vitro SARS-CoV-2 infection experiments on primary human lung epithelial cells to confirm that transcriptional upregulation of tissue factor, the extrinsic coagulation cascade master regulator, manifested at the protein level. Furthermore, infection of normal human bronchial epithelial cells with influenza A virus did not drive key regulators of blood coagulation in a similar manner as SARS-CoV-2. In addition, peripheral blood mononuclear cells did not differentially express genes regulating the extrinsic coagulation cascade or plasminogen activation system during SARS-CoV-2 infection, suggesting that they are not directly inducing coagulopathy through these pathways. The hyperactivation of the extrinsic blood coagulation cascade and the suppression of the plasminogen activation system in SARS-CoV-2–infected epithelial cells may drive diverse coagulopathies in the lung and distal organ systems. Understanding how hosts drive such transcriptional changes with SARS-CoV-2 infection may enable the design of host-directed therapeutic strategies to treat COVID-19 and other coronaviruses inducing hypercoagulation.

Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium identifies target cells, alterations in gene expression, and cell state changes.

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host–viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air–liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway.

SARS-CoV-2 infection of human iPSC-derived cardiac cells reflects cytopathic features in hearts of patients with COVID-19.

Although coronavirus disease 2019 (COVID-19) causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human induced pluripotent stem cell (iPSC)-derived heart cells to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural genes corroborates adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and nuclear disruption. Human autopsy specimens from patients with COVID-19 reflected similar alterations, particularly sarcomeric fragmentation. These striking cytopathic features in cardiomyocytes provide insights into SARS-CoV-2-induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise concerns about the long-term consequences of COVID-19 in asymptomatic as well as severe cases.

Facile accelerated specific therapeutic (FAST) platform develops antisense\xa0therapies to counter multidrug-resistant bacteria

Multidrug-resistant (MDR) bacteria pose a grave concern to global health, which is perpetuated by a lack of new treatments and countermeasure platforms to combat outbreaks or antibiotic resistance. To address this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform that can develop effective peptide nucleic acid (PNA) therapies against MDR bacteria within a week. Our FAST platform uses a bioinformatics toolbox to design sequence-specific PNAs targeting non-traditional pathways/genes of bacteria, then performs in-situ synthesis, validation, and efficacy testing of selected PNAs. As a proof of concept, these PNAs were tested against five MDR clinical isolates: carbapenem-resistant Escherichia coli, extended-spectrum beta-lactamase Klebsiella pneumoniae, New Delhi Metallo-beta-lactamase-1 carrying Klebsiella pneumoniae, and MDR Salmonella enterica. PNAs showed significant growth inhibition for 82% of treatments, with nearly 18% of treatments leading to greater than 97% decrease. Further, these PNAs are capable of potentiating antibiotic activity in the clinical isolates despite presence of cognate resistance genes. Finally, the FAST platform offers a novel delivery approach to overcome limited transport of PNAs into mammalian cells by repurposing the bacterial Type III secretion system in conjunction with a kill switch that is effective at eliminating 99.6% of an intracellular Salmonella infection in human epithelial cells.

Structural and functional study of Legionella pneumophila effector RavA.

Legionella pneumophila is an intracellular pathogen that causes Legionnaire's disease in humans. This bacterium can be found in freshwater environments as a free-living organism, but it is also an intracellular parasite of protozoa. Human infection occurs when inhaled aerosolized pathogen comes into contact with the alveolar mucosa and replicates in alveolar macrophages. Legionella enters the host cell by phagocytosis and redirects the Legionella-containing phagosomes from the phagocytic maturation pathway. These nascent phagosomes fuse with ER-derived secretory vesicles and membranes forming the Legionella-containing vacuole. Legionella subverts many host cellular processes by secreting over 300 effector proteins into the host cell via the Dot/Icm type IV secretion system. The cellular function for many Dot/Icm effectors is still unknown. Here, we present a structural and functional study of L. pneumophila effector RavA (Lpg0008). Structural analysis revealed that the RavA consists of four ~85 residue long α-helical domains with similar folds, which show only a low level of structural similarity to other protein domains. The ~90 residues long C-terminal segment is predicted to be natively unfolded. We show that during L. pneumophila infection of human cells, RavA localizes to the Golgi apparatus and to the plasma membrane. The same localization is observed when RavA is expressed in human cells. The localization signal resides within the C-terminal sequence C409 WTSFCGLF417 . Yeast-two-hybrid screen using RavA as bait identified RAB11A as a potential binding partner. RavA is present in L. pneumophila strains but only distant homologs are found in other Legionella species, where the number of repeats varies.

A Comparative Transcriptome Analysis of Human and Porcine Choroid Plexus Cells in Response to Streptococcus suis Serotype 2 Infection Points to a Role of Hypoxia.

Streptococcus suis (S. suis) is an important opportunistic pathogen, which can cause septicemia and meningitis in pigs and humans. Previous in vivo observations in S. suis-infected pigs revealed lesions at the choroid plexus (CP). In vitro experiments with primary porcine CP epithelial cells (PCPEC) and human CP epithelial papilloma (HIBCPP) cells demonstrated that S. suis can invade and traverse the CP epithelium, and that the CP contributes to the inflammatory response via cytokine expression. Here, next generation sequencing (RNA-seq) was used to compare global transcriptome profiles of PCPEC and HIBCPP cells challenged with S. suis serotype (ST) 2 infected in vitro, and of pigs infected in vivo. Identified differentially expressed genes (DEGs) were, amongst others, involved in inflammatory responses and hypoxia. The RNA-seq data were validated via quantitative PCR of selected DEGs. Employing Gene Set Enrichment Analysis (GSEA), 18, 28, and 21 enriched hallmark gene sets (GSs) were identified for infected HIBCPP cells, PCPEC, and in the CP of pigs suffering from S. suis ST2 meningitis, respectively, of which eight GSs overlapped between the three different sample sets. The majority of these GSs are involved in cellular signaling and pathways, immune response, and development, including inflammatory response and hypoxia. In contrast, suppressed GSs observed during in vitro and in vivo S. suis ST2 infections included those, which were involved in cellular proliferation and metabolic processes. This study suggests that similar cellular processes occur in infected human and porcine CP epithelial cells, especially in terms of inflammatory response.

Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro.

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40–80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.

Structure-Based Virtual Screening: Identification of a Novel NS2B-NS3 Protease Inhibitor with Potent Antiviral Activity against Zika and Dengue Viruses.

Zika virus (ZIKV), which is associated with severe diseases in humans, has spread rapidly and globally since its emergence. ZIKV and dengue virus (DENV) are closely related, and antibody-dependent enhancement (ADE) of infection between cocirculating ZIKV and DENV may exacerbate disease. Despite these serious threats, there are currently no approved antiviral drugs against ZIKV and DENV. The NS2B-NS3 viral protease is an attractive antiviral target because it plays a pivotal role in polyprotein cleavage, which is required for viral replication. Thus, we sought to identify novel inhibitors of the NS2B-NS3 protease. To that aim, we performed structure-based virtual screening using 467,000 structurally diverse chemical compounds. Then, a fluorescence-based protease inhibition assay was used to test whether the selected candidates inhibited ZIKV protease activity. Among the 123 candidate inhibitors selected from virtual screening, compound 1 significantly inhibited ZIKV NS2B-NS3 protease activity in vitro. In addition, compound 1 effectively inhibited ZIKV and DENV infection of human cells. Molecular docking analysis suggested that compound 1 binds to the NS2B-NS3 protease of ZIKV and DENV. Thus, compound 1 could be used as a new therapeutic option for the development of more potent antiviral drugs against both ZIKV and DENV, reducing the risks of ADE.

Binding mode of SARS-CoV-2 fusion peptide to human cellular membrane

Infection of human cells by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) relies on its binding to a specific receptor and subsequent fusion of the viral and host cell membranes. The fusion peptide (FP), a short peptide segment in the spike protein, plays a central role in the initial penetration of the virus into the host cell membrane, followed by the fusion of the two membranes. Here, we use an array of molecular dynamics simulations that take advantage of the highly mobile membrane mimetic model to investigate the interaction of the SARS-CoV2 FP with a lipid bilayer representing mammalian cellular membranes at an atomic level and to characterize the membrane-bound form of the peptide. Six independent systems were generated by changing the initial positioning and orientation of the FP with respect to the membrane, and each system was simulated in five independent replicas, each for 300 ns. In 73% of the simulations, the FP reaches a stable, membrane-bound configuration, in which the peptide deeply penetrated into the membrane. Clustering of the results reveals three major membrane-binding modes (binding modes 1–3), in which binding mode 1 populates over half of the data points. Taking into account the sequence conservation among the viral FPs and the results of mutagenesis studies establishing the role of specific residues in the helical portion of the FP in membrane association, the significant depth of penetration of the whole peptide, and the dense population of the respective cluster, we propose that the most deeply inserted membrane-bound form (binding mode 1) represents more closely the biologically relevant form. Analysis of FP-lipid interactions shows the involvement of specific residues, previously described as the “fusion-active core residues,” in membrane binding. Taken together, the results shed light on a key step involved in SARS-CoV2 infection, with potential implications in designing novel inhibitors.

High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2.

Heterologous polyclonal antibodies might represent an alternative to the use of convalescent plasma or monoclonal antibodies (mAbs) in coronavirus disease (COVID‐19) by targeting multiple antigen epitopes. However, heterologous antibodies trigger human natural xenogeneic antibody responses particularly directed against animal‐type carbohydrates, mainly the N‐glycolyl form of the neuraminic acid (Neu5Gc) and the α1,3‐galactose, potentially leading to serum sickness or allergy. Here, we immunized cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase and α1,3‐galactosyl‐transferase (GGTA1) double KO pigs with the Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike receptor binding domain to produce glyco‐humanized polyclonal neutralizing antibodies lacking Neu5Gc and α1,3‐galactose epitopes. Animals rapidly developed a hyperimmune response with anti‐SARS‐CoV‐2 end‐titers binding dilutions over one to a million and end‐titers neutralizing dilutions of 1:10 000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV‐19, neutralized spike/angiotensin converting enzyme‐2 interaction at a concentration <1 μg/mL, and inhibited infection of human cells by SARS‐CoV‐2 in cytopathic assays. We also found that pig GH‐pAb Fc domains fail to interact with human Fc receptors, thereby avoiding macrophage‐dependent exacerbated inflammatory responses and a possible antibody‐dependent enhancement. These data and the accumulating safety advantages of using GH‐pAbs in humans warrant clinical assessment of XAV‐19 against COVID‐19.

Antiviral Activity and Synergy of Herba Andrographidis and Radix Eleutherococci Preparations against SARS-CoV-2 Infected Vero E6 Human Primary Embryonic Kidney Epithelial Cells

Our study for the first time, demonstrates that ES, AP, and their combinations significantly inhibits SARS-CoV-2 viral growth in Vero E6 cells in a dose-dependent manner. The antiviral activity of the combinations of ES and AP are greater than expected.

SARS-CoV-2 Protease-Dependent Human Cell Infection Induces Immunological Response Imbalance and Neutrophils “Proteolytic Storm”. Proteolysis Inhibitors can Prevent and Treat Covid-19

Based on Chinese CDCP report on COVID-19, 14% of patients presented severe disease and 5% critical conditions. The average case-fatality rate was 2.3%, but mortality was as high as 49% in patients with critical illness. Serious life threatening thromboembolic complications have been found in 71•4% of non survivors and micro/macro angiopathic coagulopathy has been found, at autopsy also, with highly increased neutrophil number, fibrinogen, concentrations of D-dimer and FDPs and NETs, ATIII decrease and normal number of platelets. A cytokine storm and interaction between inflammation and coagulation has been advocated as explanation of hypercoagulability. It has been shown that SARS-CoV-2 infection of alveolar cells is driven by the S-protein by engaging ACE2 and TMPRSS2 cell receptors. whose activation depends on the activity of various host proteases. Full inhibition of SARS-CoV-2 entry was observed when serine proteases inhibitor camostat mesylate was coupled with Cathepsin B/L inhibitor E-64d. In addition multiple proteases are involved in host immune response against viral invasion and immunopathology related to imbalanced immune activation. In this paper it’s hypothesised that the severity of Covid-19 is induced by recruitment of innate responder neutrophils, which release proteases and NETs inducing endothelial damage and imbalance of the four major proteolytic cascades (coagulation, complement, fibrinolysis and kallikrein) with prevalence of activators over inhibitors and consequent thrombotic complications. Platelets adhesion to damaged endothelium and vWFVIII multimers presence, due to loss of ADAMTS13, contributes to hypercoagulability state. Human plasma or serine protease inhibitors like aprotinin can help to control neutrophil induced “proteolytic storm”. The goal of this paper is to support the view that, in SARS-CoV-2 infection, proteases have a key role and exceeding imbalanced neutrophil innate “unfriendly fire” response can be identified as the trigger of a “proteolytic storm”, responsible for subsequent well known hypercoagulation and “cytokine storm” and human plasma, in adequate volumes, together with serine proteases inhibitors can be an effective therapeutic strategy.

Transcriptomic profiling and genomic mutational analysis of Human coronavirus (HCoV)-229E -infected human cells.

Human coronaviruses (HCoVs) cause mild to severe respiratory infection. Most of the common cold illnesses are caused by one of four HCoVs, namely HCoV-229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43. Several studies have applied global transcriptomic methods to understand host responses to HCoV infection, with most studies focusing on the pandemic severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV) and the newly emerging SARS-CoV-2. In this study, Next Generation Sequencing was used to gain new insights into cellular transcriptomic changes elicited by alphacoronavirus HCoV-229E. HCoV-229E-infected MRC-5 cells showed marked downregulation of superpathway of cholesterol biosynthesis and eIF2 signaling pathways. Moreover, upregulation of cyclins, cell cycle control of chromosomal replication, and the role of BRCA1 in DNA damage response, alongside downregulation of the cell cycle G1/S checkpoint, suggest that HCoV-229E may favors S phase for viral infection. Intriguingly, a significant portion of key factors of cell innate immunity, interferon-stimulated genes (ISGs) and other transcripts of early antiviral response genes were downregulated early in HCoV-229E infection. On the other hand, early upregulation of the antiviral response factor Apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B) was observed. APOBEC3B cytidine deaminase signature (C-to-T) was previously observed in genomic analysis of SARS-CoV-2 but not HCoV-229E. Higher levels of C-to-T mutations were found in countries with high mortality rates caused by SARS-CoV-2. APOBEC activity could be a marker for new emerging CoVs. This study will enhance our understanding of commonly circulating HCoVs and hopefully provide critical information about still-emerging coronaviruses.